We are studying signaling networks in the retinal pigment epithelium (RPE) with special emphasis on lipid and retinoid metabolism pathways, differentiation/dedifferentiation pathways, and protection against oxidative or inflammatory stress. The role of signaling pathways in RPE differentiation and de-differentiation is an important focus of our research. Divergence from or convergence to the phenotype of native RPE is a common theme of much RPE cell culture research and this has an important impact on the potential use of RPE cells in cell therapy for retinal degenerations. In addition, given the likely importance of noncoding RNAs (including microRNAs (miRNAs) and long noncoding RNAs (lncRNAs)) as regulators of gene expression in the response of RPE cells to various signals, we are interested in determining changes in microRNAs (miRNAs) and long noncoding RNAs (lncRNAs)) expression in RPE cells during differentiation, and due to agents with which they are treated in our experiments. In the past year we have made progress in the following areas: 1) In this reporting period we completed a study to investigate whether IFN-, TNF- and IL-1 have any adverse effect on the expression of genes essential for RPE function, employing RPE cell line ARPE-19 as a model system. Proinflammatory cytokines IFN-, TNF- and IL-1 secreted by infiltrating lymphocytes or macrophages may play a role in triggering retinal pigment epithelial (RPE) dysfunction associated with age-related macular degeneration (AMD). Binding of these proinflammatory cytokines to their specific receptors residing on the RPE cell surface can activate signaling pathways, which in turn may dysregulate cellular gene expression. ARPE-19 cells were cultured for 3-4 months until they exhibited epithelial morphology and expressed mRNAs for visual cycle genes. We found that proinflammatory cytokines (IFN- + TNF- + IL-1) greatly increased the expression of chemokines and cytokines in these cells. However, this response was accompanied by markedly decreased expression of genes important for RPE function such as CDH1, RPE65, RDH5, RDH10, TYR and MERTK. This was associated with decreased expression of other genes highly expressed in RPE, MITF, TRPM1 and TRPM3, as well as microRNAs mir-204 and mir-211, which are known to regulate RPE-specific gene expression. The decreased expression of epithelial marker gene CDH1 was associated with increased expression of mesenchymal marker genes (CDH2, VIM and CCND1) and epithelial-mesenchymal transition (EMT) promoting transcription factor genes (ZEB1 and SNAI1). Thus, RPE cells exposed to proinflammatory cytokines IFN-, TNF- and IL-1 showed decreased expression of key genes involved in visual cycle, epithelial morphology and phagocytosis. This adverse effect of proinflammatory cytokines, which could be secreted by infiltrating lymphocytes or macrophages, on the expression of genes indispensable for RPE function may contribute to the RPE dysfunction implicated in AMD pathology. A manuscript describing these results was submitted for publication and, late in the reporting period, has been accepted. 2) We continued a study to understand the mechanisms underlying dedifferentiation of RPE cells in primary culture and re-differentiation in the ARPE-19 RPE cell line. Divergence from or convergence to the phenotype of native RPE is a common theme of much RPE cell culture research. Induced pluripotent stem (iPS) cells can be differentiated into cells sharing many aspects of RPE phenotype, and by rigorous culture methods, fetal RPE cells can be differentiated to retain or acquire aspects of native phenotype. On the other hand, explanted native RPE cells will lose important aspects of their RPE phenotype after a short time in culture. We are particularly interested in the long-known but poorly understood loss by immortalized and by primary RPE cells of expression of visual cycle enzymes. Understanding the mechanism underlying this down-regulation could be useful in ensuring that iPS-derived cells for human transplant are fully competent to restore RPE function in treated eyes. Though ARPE-19 provides a dependable and widely-used alternative to native RPE, native RPE phenotype is more and more lost with succeeding passages. Compounding this problem is the widespread use of ARPE-19 cells in an undifferentiated state. We wish to determine if suitable culture conditions and differentiation can restore RPE-appropriate expression of genes and proteins to ARPE-19, along with a functional and morphological phenotype resembling native RPE. We compared the transcriptome of ARPE-19 cells kept in long-term culture with those of native and stem-cell derived human RPE cells to assess the formers inherent plasticity relative to the latter. For these experiments, ARPE-19 cells were used at low (9-12) passage number, grown in DMEM containing high glucose and pyruvate with 1% fetal bovine serum, and differentiated for up to 4 months. We found that 4-mo ARPE-19 cells developed the classic native RPE phenotype with heavy pigmentation and RPE expressed genes including RPE65, RDH5 and RDH10, as well as miR-204/211 were greatly increased. RNA-Seq was used to assess gene expression in the differentiated ARPE-19 cells. Of 16,757 genes with detectable signals, nearly 1681 were upregulated, and 1629 were down-regulated with a fold change of 2.5 or more differences between 4 months and 4 days of culture. Gene ontology analysis shows that upregulated genes were associated with visual cycle, phagocytosis, pigment synthesis, cell differentiation, and RPE-related transcription factors, while the majority of downregulated genes play a role in cell cycle and proliferation. Comparison with data sets on stem-cell derived cultures revealed important overall similarities in expression of signature genes. The results of this study demonstrate that ARPE-19 cells can express genes specific to native human RPE cells when appropriately cultured and differentiated and thus can provide a relevant system to study differentiated cellular functions of RPE in vitro. A manuscript describing these results was submitted for publication and, late in the reporting period, is in revision. 3) We continued a project to examine the expression of secreted proteins (secretome) and exosomes in differentiated ARPE-19 cultured in DMEM with pyruvate for 4 months and exhibiting native-like RPE phenotype. We continue to collaborate with sections in the LRCMB and with other laboratories and sections (Molecular Structure and Functional Genomics), as well as with extramural labs in the analysis (HPLC and mass spectrometry) of retinoid, lipids, and other compounds. 4) We continued and expanded a project studying noncoding RNAs, originally only involving microRNAs (miRNAs), to now also involve study of long noncoding RNAs (lncRNAs). miRNAs are well known to be involved as post-transcriptional regulators of gene expression in RPE and other cells and tissues. However, while lncRNAs are less well understood they may be no less important. lncRNAs are involved in many aspects of regulation of gene transcription, post-transcriptional regulation (splicing, etc.), epigenetic regulation, and such programmatic functions as X-inactivation. Changes in expression of certain lncRNAs are associated with diseases such as various cancers and with Alzheimers disease. It is not unreasonable to expect that there will also be changes in lncRNAs associated with eye diseases such as AMD. Using RNAseq datasets we have identified lncRNAs that show changes (both up- and down-regulation) in our ARPE-19 differentiation model. Manipulation of expression of these lncRNAs results in changes to expression of a variety of important protein-coding genes. A poster describing these results will be presented at a conference late in this reporting period.